The complete mitochondrial genome sequencing of the Asian lesser white-toothed shrew (Crocidura shantungensis Miller, 1901; Eulipotyphla: Soricidae) from Jeju of Korea

Introduction

The Crocidura shantungensis (Asian lesser white-toothed shrew) Miller (1901) is classified under order Eulipotyphla, family Soricidae and genus Crocidura. The taxa that was considered C. suaveolens in East Asia was phylogenetically separated from C. suaveolens inhabiting Europe and is now treated as a separate species, C. shantungensis (Dubey et al. 2006, Jiang and Hoffmann 2001, Motokawa et al. 2003, Ohdachi et al. 2004). C. shantungensis is listed as 'Least Concern' on the IUCN (International Union for Conservation of Nature) Red List and the China Red List (Hutterer 2005, Jiang et al. 2016, Temple 2016). The Asian lesser white-toothed shrew is endemic to East Asia including Korea, Taiwan, China, Russia, Tsushima Island of Japan, and Mongolia (Hutterer 2005, Jiang and Hoffmann 2001, Motokawa et al. 2005). It is the smallest species of white-toothed shrew in South Korea and is widely distributed on the South Korean mainland as well as in its southern peripheral islands and Jeju Island (Yoon et al. 2004).

Methods

The voucher specimen was from Seogwipo-si (33°26'21.4"N 126°49'24.2"E) in Jeju Special Self-Governing Province. Voucher specimen was deposited at the National Science Museum (2021, Daejeon, South Korea. Korean Natural History Research Information System [NARIS, https://www.naris.go.kr/main.do], Hyunjong Kim and ttliu@nsmk.kr) under the voucher number NSMK-OB-MM-210300881. Our research falls under the study using a part of dead organism or animal tissue, so it does not need to undergo deliberation and approval from the committee [IACUC Standard Operating Guidelines (Revised on Dec. 2020)].

The total genomic DNA was extracted from tissues of C. shantungensis using the QIAGEN blood and tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. The amplified mitochondrial DNA was prepared to sequencing library using QIAseq FX single cell DNA library kit (QIAGEN), and DNA library was sequenced by HiseqX platform (Illumina, San Diego, CA, USA) in GnC Bio Co. (Daejeon, South Korea). The sequence assembly and gene arrangement were performed by Geneious Prime^® 2022.0.2 (Biomatters, Auckland, New Zealand).

The nucleotide dataset for phylogenetic analysis included 13 protein coding genes (PCGs) of 33 Soricidae species. Two Muridae species (Apodemus agrarius: JN629047, A. peninsulae: HQ660074) was used as outgroup. The maximum-likelihood analysis with 1000 replication of rapid bootstrapping options was performed by PhyML version 3.1 with GTR+I+G model (Guindon et al. 2010).

Results

The mitochondrial complete genome sequences of C. shantungensis (NCBI. No. OM038325) was 17,160 bp in length and contains consists of 13 protein coding genes (PCGs), 22 transfer RNA, two ribosomal RNA, one replication origin region (OL), and one control region (D- loop). Total length of the 13 protein coding genes is 11,344 bp long, all of which are encoded on the same strand except for ND6 in the light strand. The gene arrangement was exactly in accordance with other Soricidae species. The 9 PCGs (ND1, COX1, COX2, ATP8, ATP6, COX3, ND4L, ND4, ND6, CYTB) were started with initiation codon ‘ATG’, and the rest of three PCGs (ND2, ND3, ND5) were ‘ATT’. The three PCGs (COX3, ND3, and ND4) have incomplete stop codon, ‘T—’. The complete stop codon ‘TAA’ were showed in eight PCGs (ND1, COX1, COX2, ATP8, ATP6, ND4L, ND5, ND6). Also, ND2 and CYTB were terminated ‘TAG’ and ‘AGA’, respectively. The base nucleotide composition of the mitochondrial genome of C. shantungensis from Jeju in Korea is A: 32.5%, T: 32.6%, C: 21.7%, and G: 13.1% with a strong bias toward A+T (65.1%).

In the phylogenetic analysis result, the Crocidura species formed a solid monophyletic group in the family Soricidae. The result showed that C. shantungensis was well grouped with sister group to C. sibicica within the Genus Crocidura clade (Figure 1). Our result is consistent with the previous study by Jiang et al. (2019) and Li et al. (2021). The C. shantungensis from Jeju in Korea used in this study differed from C. shantungensis from other specimen from Korea, from GenBank accession number JX968507 (location is Odaesan National Park in South Korea, Kim et al. 2013) by 91 SNPs, from GenBank accession number KR560064 (exact location is unknown, expect Korea; unpublished) by 18 SNPs and three indels (two 1 bp and one 2 bp in the length). There are many reports of morphological and genetic differences between Jeju Island and the inland in various biota of Korea, and the biota of Jeju Island is sometimes classified into subspecies or species (Kim et al. 2018, Komiya 1985, Lee and Oh 2021, Oh et al. 2012). The C. shantungensis is distributed throughout the Korean Peninsula, and we presented the complete nucleotide sequence of the mitochondrial genome for the C. shantungensis, which is distributed on Jeju in Korea. We reported genetic differences from JX968507 (location is Odaesan National Park in South Korea, Kim et al. 2013). These findings are useful for inferring biogeographic and phylogenetic studies within the Soricidae family, and especially for the crocidura genus. The genomic resources of Jeju in Korea obtained through this study can be used as reference data for genetic research as well as the phylogenetic relationship and evolutionary history of Crocidura species.

Figure 1. Maximum-likelihood (ML) tree based on analysis of 13 protein-coding genes of 33 Soricidae species.

Numbers on branches represent bootstrap values.

Data availability